Background: TeamSTEPPS (Team Strategies and Tools to Enhance Performance and Patient Safety) is a team-training intervention which shows promise in aiding the mitigation of medical errors. This article examines the construct validity of the TeamSTEPPS Teamwork Perceptions Questionnaire (T-TPQ), a self-report survey that examines multiple dimensions of perceptions of teamwork within healthcare settings. Method: Using survey-based methods, 1700 multidisciplinary healthcare professionals and support staff were measured on their perceptions of teamwork. Confirmatory factor analysis was conducted to examine the relationship between the five TeamSTEPPS dimensions: Leadership, Mutual Support, Situation Monitoring, Communication, and Team Structure. Results: The analysis indicated that the T-TPQ measure is more reliable than previously thought (Cronbach's α=0.978). Further, our final tested model showed a good fit with the data (x2 (df) 3601.27 (546), p<0.0001, Tucker–Lewis Index (TLI)=0.942, Comparative fit index (CFI)=0.947, root mean square error of approximation (RMSEA)=0.057), indicating that the measure appears to have construct validity. Further, all dimensions correlated with one another, but were shown to be independent constructs. Conclusions: The T-TPQ is a construct-valid instrument for measuring perceptions of teamwork. This has beneficial implications for patient safety and future research that studies medical teamwork.